A continued study of site-specifically modified RNAs is proposed in which a combination of synthetic and biophysical approaches will be taken. The long-term goal of the work is to understand the functional significance of modified nucleosides in RNA and their roles in regulation of RNA interactions. The design of new RNA-targeting drugs or RNA-based therapeutics will ultimately rely on a fundamental understanding of RNA structure-function relationships. The short-term goal is to obtain detailed information regarding the roles of natural modified bases such as pseudouridine and their contributions to RNA structure and stability. Modified RNAs that will be synthesized and structurally characterized are an exposed hairpin in domain IV of E. coli 23S rRNA and the analogous large subunit rRNA hairpin in eukaryotes. These RNAs contain highly conserved pseudouridine modifications and nucleotide sequences that have been shown to play a role in ribosome assembly. Results of the proposed structure studies will be compared to in rive functional studies. The specific aims of the research plan are as follows: 1) the biophysical properties and three-dimensional structures of modified 19-nt RNA hairpins from helix 69 (residues 1906-1924) of E. coli 23S rRNA will be determined 2) the structure and properties of a fully modified 19-nt human analogue of the 1920-region RNA from E. coli will be determined, 3) the structures of the 19-nt, fully modified 1920-region RNA constructs and full-length, biologically relevant, large subunit rRNAs will be compared by reactions with transition metal complexes and organic reagents, and 4) ligands that have specificity for the bacterial 1920-region RNA structure will be identified and used to study the 1920 RNA function. Taken together, the proposed experiments will be used to determine whether modified bases such as pseudouridine or 3-methylpseudouridine influence local RNA structure enough to provide suitable recognition elements for selective drug targeting.